Despite the recent progress, each day approximately 1,600 children become HIV infected, the vast majority of these infections occur in Africa. One-third to one-half of these perinatal HIV infections is due to breast-feeding. Formula feeding is not an option due to economic and cultural imperatives, and because it is often not safe in environments with out access to clean water. For the near term, an enhanced understanding of the factors that contribute to breast milk transmission is essential to help devise appropriate and sustainable efforts to minimize MTCT of HIV in developing countries. The role that potential determinants play on breast milk transmission remains unclear. This proposal will employ a large and well-organized cohort of breast-feeding HIV infected women, the Zambian Exclusive Breast-feeding Study (ZEBS). We will characterize important aspects of HIV breast milk transmission with respect to the virologic and immunologic constitution of breast milk and with respect to the role of local inflammatory disease. The first specific aim of this proposal is to characterize HIV variants in breast milk. We hypothesize that the breast milk is a distinct virologic compartment. We will use HTA and clonal sequencing to establish prevalence and source (i.e. plasma vs.cells) of viral co-mingling between these compartments. Differentially expressed bands will be identified, cloned and sequenced. The second aim is determine the effects of mastitis on the HIV quasispecies. Substantial evidence points to an important role oft mastitis in enhancing HIV transmission. Few data exist on the mechanism of this effect on transmission risk. Does it simply increase the population of existing variants or does it produce novel variants that are less subject to immune clearance? We hypothesize that inflammatory cytokines reactivate latent ancestral variants and increase local production. This not only increases viral diversity, resulting a higher probability of a "transmissible strain" but also may select for variants that have escaped ongoing immune surveillance. We will use HMA/HTA as well as clonal sequencing and phylogenetic analysis to define the changes induced by inflammation. The final aim is characterize HIV specific T cell responses in lactating women. We hypothesize that, as in blood, CTL responses play a critical role in HIV suppression. We will assess breast milk CTL response in comparison to blood. These data will be correlated with levels of HIV in both compartments. These studies will provide the foundation for further studies correlating CTL and MTCT and will provide important immune correlates of MTCT.